Project summary: RNA granules are membraneless RNA-protein condensates proposed to form by liquid- liquid phase separation (LLPS). LLPS is a spontaneous process in which molecules transistion between dilute and condensed liquid phases. The liquid properties of membraneless condensates were first characterized for P granules, the germ granules of C. elegans. However, the mechanism of assembly of P granules in the germline of C. elegans is unclear. My initial findings have demonstrated that the liquid phase of P granules is not stable on its own and requires a gel-like scaffold to assemble in the cytoplasm. Emerging evidence has suggested that other RNA granules (such as stress granules) also contain multiple phases, including phases that are non-dynamic. The significance of these solid phases, however, is not clear. RNA granule proteins that form solid phases are generally associated with pathology in diseases associated with aging, and it is thought that they do not play a role in normal granule assembly. My goal is to use a combination of biochemical and genetic experiments to explore the molecular interactions and biophysical principles that drive P granule assembly through a gel-like scaffold. The gel-like scaffold of P granules is made by MEG-3 and its paralog MEG-4. The MEG proteins are intrinsically disordered proteins related to the GCNA family of disordered proteins conserved throughout eukarya, from plants to humans1. GCNA proteins have been shown to localize to P granules in C. elegans, and intriguingly to form condensates in human cancer cells. Gel-like condensates may, therefore be a common feature of eukaryotic cells. We hypothesize that gel-like phases are critical for the assembly and localization of liquid phases. In Aim 1 of this proposal, I will develop an in vitro system to reconstitute multi- phase P granules using purified components to investigate the role of the gel-like MEG-3 phase in condensate assembly. I will determine the regions of MEG-3 required for forming a gel-like phase and develop a model for the role of gel-like phases in granule assembly both in vitro and in vivo. In Aim 2, I will investigate the GCNA family of germline proteins using both C. elegans and human cell models. Using techniques developed in Aim 1, I will address if GCNA family proteins form gel-like scaffolds in membraneless condensates and investigate the role of GCNA in the germline.